The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography

Abstract

The Qbeta host factor, a heat-stable protein necessary in concert with Qbeta replicase for phage Qbeta RNA replication in vitro, has been localized in Escherichia coli and found to be associated primarily with ribosomes. This location has been established both by complement fixation assays with highly specific antiserum directed against the host factor, and by in vitro stimulation of Qbeta RNA replication by the Qbeta replicase. The complement fixation assay has provided the estimate that there are approximately 2500 copies of the host factor polypeptide per cell. The host factor is released from the ribosomes by a 1 M NH4Cl wash and concentrated by ammonium sulfate precipitation. It can be purified to apparent homogeneity in one further step by chromatography on poly(A)-cellulose. Ribosomal protein S1 subunit I of Qbeta replicase) also binds to the poly(A)-cellulose column and elutes before the host factor. In agreement with previous reports, we find that the host factor has a monomer molecular weight of 12,000 as judged by sodium dodecyl sulfate-polyacrylamide gels, and a native molecular weight of 72,000 as judged by the stoichiometric interaction of the host factor with Qbeta RNA, by sedimentation in sucrose velocity gradients, and by sodium dodecyl sulfate gel mobility when incompletely disaggregated. The Qbeta host factor is a potent inhibitor of an in vitro poly(A)-directed polylysine protein-synthesizing system, but has less effect on the in vitro translation of poly(U), R17 RNA, late T7 mRNA, or endogenous E. coli mRNA. The amino acid composition and NH2- terminal sequence rule out the host factor as one of the known 30 S or 50 S E. coli ribosomal proteins. The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with ribosomes or are involved in the protein-synthetic machinery of the cell.